Method for the preparation of cholesterol oxidase

ABSTRACT

A method for preparing cholesterol oxidase, comprising the steps of: 
     (a) growing a cholesterol oxidase producing microorganism in a medium comprising a nontoxic concentration of nonionic surfactant to yield extracellular cholesterol oxidase; and 
     (b) separating the extracellular cholesterol oxidase from the medium. 
     Preferably, cholesterol oxidase is prepared from a growth medium of Nocardia cholesterolicum species NRRL 5767 or 5768 which contains cholesterol, a suitable derivative of cholesterol or a 3-β-hydroxy sterol as a cholesterol oxidase inducer and a nonionic surfactant in a concentration which is not toxic to said bacterium, which does not produce decomposition products in concentrations sufficient to be toxic to said bacterium, and in sufficient concentration to produce extracellular enzyme.

This is a division of application Ser. No. 629,955, filed Nov. 7, 1975,now U.S. Pat. No. 4,035,237.

FIELD OF THE INVENTION

The present invention relates to an improved method for the synthesis ofcholesterol oxidase and more particularly to an improved method for thesynthesis of extracellular cholesterol oxidase.

BACKGROUND OF THE INVENTION

Microorganisms capable of metabolizing cholesterol are potential sourcesof enzymes useful in an enzymatic assay of cholesterol is complexmixtures such as blood serum, etc. This is particularly so if themicroorganisms can use cholesterol as a sole carbon source, for in thisassay process cholesterol must be degraded by oxidative enzymes.

Stadtman, T. C., Methods in Enzymology, Vol. 1, Colowick, S. P. andKaplan, N. O., Eds. Academic Press, N.Y. 1955, P. 678 and Stadtman, T.C., Cherkes, A. and Anfinsen, J., Biol. Chem., 206, 510 (1954) reportedthe preliminary purification of an enzyme from Nocardia cholesterolicum,an organism originally isolated by Schatz et al (Schatz, A., Savard, K.,and Pintner, I. J., J. Bacteriol., 58, 117-125 (1949). Stadtman'senzyme, "cholesterol dehydrogenase," was purified sufficiently for usein a cholesterol assay based on the measurement of the increase inabsorbance at 250 nm owing to the formation of cholest-4-en-3-one. Sinceas we have now determined, the direct acceptor of cholesterol electronsin this oxidation is oxygen, the enzyme should properly be calledcholesterol oxidase according to current convention.

The bacterial strains described by Stadtman when cultured as describedin the aforementioned references produce very low enzyme levels whichare not practical for commercial operations. These levels aresufficiently low that purification of the enzyme is a very remotepossibility for achieving a commercial operation.

Goodhue et al in U.S. Pat. No. 3,909,359 issued Sept. 30, 1975, describean improved method for the production of the Stadtman cholesteroloxidase which comprises the steps of:

A. GROWING THE BACTERIUM Nocardia cholesterolicum species NRRL 5767 orNRRL 5768 in a medium in which cholesterol or a suitable derivativethereof serves as an auxiliary source of carbon; and

B. ISOLATING FROM SAID MEDIUM A CELL-FREE EXTRACT CONTAINING THE ACTIVEENZYME.

Although the method described in this application is greatly improvedover the original synthesis described by Stadtman and can be said torender the process commercially practical, it still poses a shortcomingin that the enzyme is produced predominantly intracellularly. For thisreason actual extraction of the enzyme from the growth medium requiresthe use of time-consuming, expensive cell disruption techniques such ashomogenization, etc.

German OLS 2,246,695 published Mar. 29, 1973, describes a method ofisolating a cholesterol oxidase enzyme produced by a culture of Nocardiamicroorganism identified as NRRL 5635 and NRRL 5636. According to themethod described therein, the harvested cells are treated with anonionic surfactant and stirring at room temperature to release a largeproportion of the enzyme from the cells into the supernatant therebyeliminating the need for involved cell extraction and isolationtechniques. Using this technique for enzyme extraction we havecalculated that yields on the order of about 40 to 160 U per liter areobtined.

Reese, E. T. and Maquire, A., in Surfactants as Stimulants of EnzymeProduction by Microorganisms, Applied Microbiology, February, 1969, P.242 - 245 describe the observation that the addition of sorbitanpolyoxyethylene monooleate (Tween 80 from Atlas Chemical Co.,Wilmington, Delaware) and other nonionic surfactants to fungal cultureswhich normally produce extracellular enzymes, results in a markedincrease in enzyme yield.

British Pat. No. 1,385,319 describes a process for producing cholesteroloxidase from Nocardia species NRRL 5635 and NRRL 5636. During thefermentation, a suspension of cholesterol is slowly added as an inducerfor cholesterol oxidase. A non-ionic surfactant, Tween 80, at a level of3% by volume is used to disperse the cholesterol in the suspension. Thisresults in only a minute amount of non-ionic surfactant in thefermentation medium, which amount is apparently insufficient to produceextracellular enzyme.

The bacterial cultures known to produce cholesterol oxidase, normallyproduce the enzyme intracellularly. The present invention provides animproved method for the production of cholesterol oxidase, wherein theenzyme is produced extracellularly thereby eliminating the need for celldisruption.

SUMMARY OF THE INVENTION

Attainment of the foregoing result and other advantages which willbecome apparent in the discussion which follows are obtained byincluding in the growth medium a nonionic surfactant in a concentrationwhich is itself nontoxic and whose decomposition products are not insufficient concentration to be toxic to the cholesteroloxidase-producing organism. The inclusion of this ingredient results inthe majority of the enzyme being produced extracellularly. Thus,substantial reduction in the time and cost incident to enzyme extractionafter production are achieved by eliminating the need for celldisruption which is generally required when the enzyme is producedintracellularly.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates cholesterol oxidase activity as a function offermentation time and also the depletion of cholesterol in thefermentation medium with time.

FIG. 2 illustrates the relationship between cholesterol oxidase activityand the presence of cholesterol and deoxycholate (DOC).

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention principally extracellular cholesteroloxidase production is achieved when a nonionic surfactant which isitself not toxic and whose decomposition products are nontoxic to themicroorganism is added to a growth medium which produces cholesteroloxidase. Preferably, the bacterium, Nocardia cholesterolicum speciesNRRL 5767 or NRRL 5768, is grown in a medium comprising cholesterol, asuitable derivative thereof, or a 3-β-hydroxy sterol as an inducer ofcholesterol oxidase.

According to a preferred embodiment, the nonionic surfactant has apolyoxyethylene or polyglycidol hydrophilic moiety and a lipophilicmoiety comprising at least nine carbon atoms. According to a furtherpreferred embodiment, the lipophilic moiety comprises a fatty acid chainof at least ten carbon atoms.

Any culture which produces cholesterol oxidase can be used in thepractice of this invention. Cultures known to produce cholesteroloxidase include, for example, Arthobacter crystallopoities, Arthobacterstrain NP, Corynebacterium species, Mycobacterium strain MA-7,Mycobacterium strain E-16, Mycobacterium rhodochrous, or other speciesfrom so-called Mycobacterium rhodochrous group, Mycobacterium rubrum,Nocardia erythropolis, Nocardia species NRRL 5635 and 5636, Nocardiacholesterolicum -- smooth and Nocardia cholesterolicum -- rough.

Two Nocardia cholesterolicum cultures which yield cholesterol oxidaseand are especially preferred in the practice of the present inventionare characterized as the "rough" and "smooth" strains and are calledNRRL 5767 and NRRL 5768 respectively based on their deposit with theAgricultural Collection Investigations Fermentation Laboratory, Peoria,Illinois.

Complete details of the organisms are as follows:

Description of Nocardia cholesterolicum

I. Cellular morphology.

A. Smooth strain. Gram positive weakly acid-fast, coryneform, nowell-developed mycelia, but rudimentary branching observed. Coccoidforms appear in older cultures.

B. Rough strain. Same as above.

II. Colonial morphology.

A. Nutrient agar (5 days, 30° C).

1. smooth strain. circular, convex, watery, entire, smooth, glistening,pink-white. No soluble pigment.

2. Rough strain. circular, convex, entire, smooth to rough, pink-white.No soluble pigment.

B. Yeast glucose agar (5 days, 30° C).

1. smooth strain. cream to tan-colored, watery, smooth, round, andelevated.

2. Rough strain. dry, cream to tan-colored, round, and elevated.

C. Casein agar (5 days, 30° C).

1. smooth strain. cream to tan-colored, watery, round, smooth, elevated.

2. Rough colony. Tan to pink, dry, elevated.

D. Gelatin agar.

1. Smooth strain. circular, convex, entire, smooth, watery,cream-colored.

2. Rough strain. circular, convex, entire, dry, cream-colored.

III. Growth in liquid culture (Nutrient broth, 5 days, 30° C).

A. Smooth strain. off-white to tan flock-forming precipitate, nopellicle.

B. Rough strain. off-white to tan flock-forming precipitate, nopellicle.

    ______________________________________                                        IV.   Physiology (Smooth and Rough strains identical)                         Aeration                   aerobic                                            Gelatin hydrolysis         -                                                  Casein hydrolysis          -                                                  Starch hydrolysis          -                                                  Oxidase                    -                                                  Catalase                   +                                                  Urease                     -                                                  Indole                     -                                                  Methyl red                 -                                                  Phenylalanine deamination  -                                                  Litmus milk                alkaline                                           Use of compounds as sole carbon source                                        Citrate                    +                                                  Lactate                    +                                                  Malate                     +                                                  Succinate                  +                                                  Fructose                   +                                                  Glucose                    +                                                  Sucrose                    ±                                               Maltose                    +                                                  Glycerol                   +                                                  Sorbitol                   +                                                  Trehalose                  +                                                  Raffinose                  -                                                  Dulcitol                   -                                                  Lactose                    -                                                  Mannitol                   +                                                  Starch                     -                                                  Arabinose                  -                                                  ______________________________________                                    

According to the method described in Goodhue et al, which producedintracellular cholesterol oxidase, the use of a conventional primarycarbon source such as glycerol, in combination with a secondary orauxiliary carbon source such as cholesterol, cholest-4-en-3-one, orcholesteryl linoleate, all of which act as cholesterol oxidase inducers,increases the yield of cholesterol oxidase enzyme to levels about 100times higher than those produced when cholesterol oxidase inducer is notused or when cholesterol is used as the sole carbon source as describedin the prior art.

Thus, according to Goodhue improved yields were obtained when thebacterium was grown in a conventional nutrient medium of the type wellknown in the art which generally comprises a nitrogen source such asammonium sulfate, a potassium and a phosphorus source such as potassiumphosphate, trace metal ions, and a mixture of a primary carbon sourcesuch as glycerol and a cholesterol oxidase inducer selected from thegroup consisting of cholesterol, cholest-4-en-3-one, cholesteryllinoleate, and mixtures thereof. The pH value of the medium ismaintained between about 5.0 and 8.0, preferably between about 6.5 and7.5, at a temperature of from about 18 to about 35° C, preferably about25°-30° C, for a period of from about 18 to about 40 hours, preferablyfrom about 20 to about 24 hours.

The quantities of nitrogen, potassium phosphate and trace metal ionsused in the culture are those conventionally used in processes of thistype and are well known to those skilled in the art. Specifically, thosedescribed in the aforementioned references provide useful levels ofthese constituents.

Among the primary carbon sources which were found useful by Goodhue etal and which are similarly useful herein are glycerol, glucose, andacetic acid. Conventional concentrations of primary carbon source areused. These generally range from about 0.5% to about 5% by weight. Theconcentration of the cholesterol oxidase inducer utilized, generallyranges from about 0.05% to about 0.5% by weight. A preferred range ofinducer is from about 0.1% to about 0.2% by weight.

According to the improved process described herein, cholesterol oxidaseis prepared substantially as described by Goodhue et al except that thegrowth medium further includes a non-inhibiting concentration of anonionic surfactant which is not toxic to the microorganism and whosedecomposition products are similarly nontoxic as described hereinafter.Inclusion of such a material in the fermentation medium results in theproduction of primarily extracellular cholesterol oxidase whicheliminates the necessity for time-consuming, complicated and costly cellharvesting extraction techniques of the sort used in the prior art.

Any nonionic surfactant which meets the aforementioned criteria isuseful in the successful practice of the instant invention.

Nonionic surfactants are well known in the art and no further definitionthereof is required herein. Typical examples of such materials includepolyethylene glycol, polyvinyl alcohol, polyethers, polyesters, andpolyhalides.

Of critical importance to the successful practice of the invention arethe criteria that

1. neither the particular nonionic surfactant used nor its decompositionproducts are toxic to the microorganism in the concentrations requiredto produce extracellular enzyme; and

2. the amount of surfactant used does not inhibit enzyme production.

Surfactant toxicity for the microorganism can, of course, be determinedreadily by subjecting a culture of the microorganism to a relatively lowconcentration of the surfactant, on the order of about 0.5 g/l, andobserving the effect of such a treatment on the microorganism.Inhibition of growth of the microorganism will generally result in zonesof retarded growth of the culture in a petri plate where contact with atoxic material occurred.

The toxicity of the decomposition products of the surfactant can betheorized as described briefly hereinafter, however, the only positivetest for such a criterion is evaluation in the growth medium andobservation of the effects of by-products produced therein as shown inthe examples below.

Although the inventors do not wish to in any way be limited to aspecific theory for the mechanism of the successful practice of theirinvention, it appears that, as demonstrated in the examples below,nonionic surfactants which are known to decompose into toxic moieties,e.g., into phenols, are not useful in the successful practice of theinvention.

Useful nonionic surfactants include a broad range of materials and anysuch material which meets the two criteria described hereinabove areuseful in the successful practice of the invention.

According to a preferred embodiment wherein cholesterol oxidase isprepared as described hereinabove the nonionic surfactant has apolyoxyethylene or polyglycidol hydrophilic moiety and a lipophilicmoiety comprising at least nine carbon atoms. According to a highlypreferred embodiment the lipophilic moiety comprises a fatty acid chainof at least ten carbon atoms.

Optimum results are achieved when the fatty acid chain contains at least16 carbon atoms and the hydrophilic moiety comprises about 20polyoxyethylene units.

The following is a list of specific surfactants found particularlyuseful in the successful practice of the instant invention and theirstructure:

    ______________________________________                                                             No. of            No. of                                 Surfactant                                                                            Hydrophile   Units   Lipophile Units                                  ______________________________________                                        S-1     Sorbitan     1       Lauric acid                                                                             1                                              Polyoxyethylene                                                                            20                                                       S-2     Sorbitan     1       Lauric acid                                                                             1                                              Polyoxyethylene                                                                            4                                                        S-3     Sorbitan     1       Palmitic acid                                                                           1                                              Polyoxyethylene                                                                            20                                                       S-4     Sorbitan     1       Stearic acid                                                                            1                                              Polyoxyethylene                                                                            20                                                       S-5     Sorbitan     1       Stearic acid                                                                            1                                              Polyoxethylene                                                                             4                                                        S-6     Sorbitan     1       Stearic acid                                                                            3                                              Polyoxyethylene                                                                            20                                                       S-7     Sorbitan     1       Oleic acid                                                                              1                                              Polyoxyethylene                                                                            20                                                       S-8     Sorbitan     1       Oleic acid                                                                              1                                              Polyoxyethylene                                                                            5                                                        S-9     Polyoxyethylene                                                                            10      Nonylphenyl                                                                             1                                      S-10    Polyoxyethylene                                                                            15      Nonylphenyl                                                                             1                                      S-11    Polyoxyethylene                                                                            30      Nonylphenyl                                                                             1                                      S-12    Polyglycidol 6       Nonylphenyl                                                                             1                                      S-13    Polyglycidol 10      Nonylphenyl                                                                             1                                      ______________________________________                                    

Among the nonionic surfactants which were not found useful in thesuccessful practice of the invention were the octylphenyl polyethoxyethanols commercially available from Rohm and Haas of Philadelphia, Pa.,under the Triton trademark. It is theorized that such materials producedtoxic phenols upon decomposition and that they were thereforeunsatisfactory. S-14 (Triton-X-100) which is of this class and comprises9.5 polyethoxyethylene units and 1 octylphenyl unit is evaluated in theExamples below.

Specific examples of each class of the foregoing compounds, theirrelative effectiveness in releasing cholesterol oxidase, commerciallyavailable materials of this type and optimum concentrations aredemonstrated and defined in the examples which follow.

The concentration of nonionic surfactant used in any particular growthmedium will vary considerably depending upon the composition of themedium, the sensitivity of the medium to the particular surfactant andthe particular surfactant used. Generally, however, surfactantconcentration ranges of from about 0.5 to about 10.0 g/liter of mediumhave been found useful in at least certain fermentation media withcertain surfactant compositions. At levels above 10 g/liter thesurfactant generally acts as a growth inhibitor, most probably due tosurfactant or surfactant by-product toxicity to the micro-organism. Atlevels below 0.5 g/liter no significant release of cholesterol oxidaseis observed. It is generally preferred, as demonstrated by the extensiveexemplary results set forth below, to utilize surfactant concentrationsbetween about 1 and 5 g/liter of medium.

As described hereinabove, the growth medium includes a primary carbonsource such as, for example, glycerol, reagent grade glucose, technicalgrade corn syrup, or the like, and an inducer of cholesterol oxidasewhich may also be an auxiliary carbon source. Besides the cholesterol,cholest-4-en-3-one and cholesteryl linoleate which were taught asinducers by Goodhue et al, supra, other sterols and cholesterol estersare useful as inducers of cholesterol oxidase. Other preferred inducersinclude, for example β-sitosterol and 5α-cholestan-3-β-ol, andcholesterol esters such as cholesteryl oleate, cholesterol linolenate,cholesteryl formate and cholesteryl propionate.

Any conventional nitrogen compound can be used as the nitrogen source inthe growth medium. An especially preferred nitrogen source for thepractice of the present invention is Nutrient Broth, a product of DifcoLaboratories, Detroit, Michigan, containing peptone and beef extract.Other nitrogen sources found useful in the practice of this inventioninclude, for example, Anatone, available from Cudahy Laboratories,Omaha, Nebraska, and N-Z/amine Type AT, N-Z/amine Type BT, N-Z/amineType ET, N-Z/amine Type TT, Soy Petone Type T, Fermamine Type I,Fermamine Type II and Fermamine Type III, all available from SheffieldChemical Div. of Kraftco Corp., Union, New Jersey.

In the production of cholsterol oxidase using a growth medium containinga nonionic surfactant in accordance with the teachings of thisinvention, foaming is often encountered. In order to control thefoaming, especially when producing large batches, use of a foam controlagent is advisable. One such foam control agent found useful in thepractice of this invention is Polyglycol P-2000, available from DowChemical Co. (Midland, Michigan). Other foams control agents can also beused, the main criteria for selection and use being the lack ofinhibition of enzyme synthesis at a concentration level which willcontrol the foam.

In the following examples which are presented to better demonstrate thesuccessful practice of the invention the following definitions apply:

1. Culture: unless otherwise stated the "rough strain" (NRRL 5768) ofNocardia cholesterolicum was used.

2. Nutrient Media

The compositions of media used in the Examples are as follows:

    ______________________________________                                        a)  Glycerol medium         per liter                                             Ammonium sulfate        2.0 g                                                 Potassium phosphate (dibasic)                                                                         2.0 g                                                 Salt solution "C"       5.0 ml                                                Glycerol                5.0 g                                                 Tryptone                0.1 g                                                 Cholesterol             1.0 g                                                 Distilled water         to 1 liter                                            Salt solution "C"       per liter of                                                                  0.1 N HCl                                             Magnesium sulfate . 7H.sub.2 O                                                                        25.0 g                                                Calcium chloride . 2H.sub.2 O                                                                          0.1 g                                                Ferrous sulfate . 7H.sub.2 O                                                                           2.8 g                                                Manganese sulfate . H.sub.2 O                                                                          1.7 g                                                Zinc sulfate . 7H.sub.2 O                                                                              0.06 g                                               Sodium chloride          0.6 g                                            b)  Modified glycerol medium                                                      Same as (a) with        per liter                                             Inositol                1.0 g                                                 Yeast extract           1.0 g                                             c)  Glucose medium                                                            Same as (a) except glycerol is replaced                                       with glucose.                                                                 d)  S-3 medium              per liter                                             Nutrient broth*         8.0 g                                                 Yeast extract           1.0 g                                                 Inositol                1.0 g                                                 Cholesterol             1.0 g                                                 S-3                     5.0 g                                                 Distilled water         to 1 liter                                        e)  Yeast extract medium    per liter                                             Yeast extract           1.0 g                                                 Inositol                1.0 g                                                 Sodium phosphate (dibasic)                                                     7H.sub.2 O             2.0 g                                                 Tryptone                5.0 g                                                 Distilled water         to 1 liter                                        f)  Inoculum medium         per liter                                             Glucose                 10.0 g                                                Yeast extract           10.0 g                                                Potassium phosphate                                                            (dibasic)               1.0 g                                                Salt solution A-1        2.0 ml                                               Salt solution A-2        2.0 ml                                               Agar                    20.0 g                                            Adjust pH to 7.0 and made up to 1 liter with                                  distilled water.                                                                  Salt solution A-1                                                                                per liter of 0.1 N HCl                                     Magnesium sulfate . 7H.sub.2 O                                                                   100.0 g                                                    Ferrous sulfate . 7H.sub.2 O                                                                      10.0 g                                                    Manganese sulfate . 7H.sub.2 O                                                                    1.0 g                                                     Sodium molybdate . 2H.sub.2 O                                                                     0.5 g                                                 Made up to 1 liter with 0.1N hydrochloric acid.                                   Salt solution A-2                                                                                     per liter                                             Calcium chloride        10.0 g                                                Deionized distilled water                                                                             to 1 liter                                        g)  Modified S-3 medium     per liter                                             Nutrient broth          8.0 g                                                 Yeast extract           1.5 g                                                 Cholesterol             2.0 g                                                 S-3                     5.0 g                                                 Polyglycol P-2000       0.3 g                                             ______________________________________                                         *A product of Difco Labs, (Detroit, Mich) containing peptone and beef         extract.                                                                 

3. Maintenance of the Culture

The cultures are maintained on the slants of glycerol medium containingcholesterol and are transferred every second day.

4. Preparation of Inoculum (Small Scale Use)

A slant of the inoculum medium (f) is inoculated with Nocardiacholesterolicum (rough) from a two-day-old glycerol medium slant andincubated at 30° C for 48 hours. The culture from this slant is removedwith a wire loop and resuspended in 25 ml of sterile distilled water byvigorous shaking. The turbidity of the suspension is generally between1.8 - 2.2 Optical Density (O.D.) units at 660 nm. Sixty ml of thissuspension is used per liter of the medium to be inoculated.

5. Preparation of Inoculum for Large Scale Fermentation

Six 2.8 liter Fernbach flasks, each containing one liter of modified S-3medium, are inoculated with 2-day-old culture of Nocardiacholesterolicum-rough grown on the slants of inoculum medium. Theprocedure used for this purpose is described above. One slant is usedper flask. The flasks are shaken at 125 RPM and 30° C for 19 hours.

6. Fermentation

The fermentations are carried out in 2.8 liter Fernbach flasks and in250 ml Erlenmeyer flasks. The volumes of medium used in the Fernbachflasks and Erlenmeyer flasks are 1 liter and 25 ml respectively. Themedium in the flasks is inoculated as described above and incubated at30° C. The shaker speed is adjusted to 125 RPM for the Fernbach flasksand to 200 RPM for the Erlenmeyer flasks. These medium volumes and theshaker speeds were selected because they resulted in similar rates ofoxygen transfer. The samples are withdrawn aseptically every 24 hoursfor the meaurement of the cholesterol oxidase activity.

7. Harvesting the Cells

The cells are harvested (i.e., separated from the fermentation broth) by15 minutes centrifugation in a refrigerated centrifuge (I. Sorvall Inc.,Norwalk, Conn.) at 12,350 × g.

8. Determination of Cholesterol Oxidase Activity

a. Preparation of cell fractions for the assay of cholesterol oxidase.

Cholesterol oxidase can be present outside the cell or extracellularlyand inside the cell or intracellularly. Further, the intracellularenzyme can be present as free or soluble enzyme and as bound orinsoluble enzyme. The extracellular enzyme can be assayed in the brothafter the removal of the cells by centrifugation. To measure theintracellular enzyme the cells are disrupted by sonication.

The cell pellet obtained by centrifugation is suspended in 1 ml ofdistilled water and diluted to 20 ml with 50 mM potassium phosphatebuffer (pH 7.0). It is sonicated for 5 minutes in an ice-water bath, in1 minute bursts at 30 second intervals. The sonicated suspensions arecentrifuged at 27,000 × g for 15 minutes in the cold. The activity inthe supernatant is called the intracellular, soluble activity. Thepellet is resuspended in 2% sodium deoxycholate and allowed to stand onice for 10 minutes. It then is centrifuged at 27,000 × g for 15 minutesin the cold. The cholesterol oxidase activity in the supernatant iscalled the intracellular, insoluble activity. The sum of theextracellular, the intracellular soluble and the intracellular insolubleactivities is called the total activity.

9. Enzyme Assay

Cholesterol oxidase activity is measured by the following technique:

Reagents:

a. 50 mM Potassium Phosphate buffer pH 7.0 (KP buffer): 30.5 ml 0.2 M K₂H PO₄ + 19.5 ml 0.2 M KH₂ PO4 + water to 200 ml final volume.

b. 0.1% dianisidine solution: 10 mg 3,3'-Dimethoxybenzidinedihydrochloride per ml water. No pH adjustment.

c. Reagent Buffer: Add 0.4 ml dianisidine solution and 1.4 mg peroxidasepowder (Sigma Type II, horseradish peroxidase, RZ 1.0 - 1.5 No. P8250)to 40 ml KP buffer, mix, dilute to 50 ml with KP buffer. The solutionwill turn turbid when the dianisidine is added but clears when mixed.This solution should be kept cold until ready to use. We have storedreagent buffer at 4° C for 3 days without problems, but routinely thisreagent is prepared fresh daily.

d. Cholesterol solution: To 10 ml Triton X-100 (S-14) heated on a hotplate add 300 mg cholesterol powder and mix with stirring rod untilsolution clears. Add 90 ml water and stir. The solution will be cloudy,now continue mixing the flask by swirling it under a stream of coldwater; the solution will become clear. Turbidity was due to detergentcoming out of solution, the cooling rehydrates the detergent and fullysolubilizes the steroid. This solution is stable for one week whenstored at room temperature. ##EQU1##

Assay:

a. 6.7 ml of reagent buffer plus 0.3 ml substrate are combined in a testtube, mixed, and placed in a waterbath set at 37° C. After five minutes1.0 ml of enzyme solution is added to give 8 ml final volume in the tubeand an initial reading at 430 nm on a Spectronic 20 spectrophotometer(Bausch and Lomb) is recorded. The tube is replaced in the waterbath.Tubes are read in the spectrophotometer every five minutes for 25minutes. Rate of color development is determined from a plot of O.D.change vs. time, by averaging the O.D. change throughout the linearportion of the curve. Activity is calculated using a constant previouslydetermined for the dye system from a standard curve. Enzyme preparationswere diluted so that 0.005 to 0.06 units of cholesterol oxidase wereused per assay tube.

b. For continuous assay of cholesterol oxidase a Beckman recordingspectrophotometer is used. In this procedure 2.5 ml reagent buffer, 0.1ml substrate and water are combined in a 3 ml cuvette. When temperatureequilibration has occurred, enzyme is added and the rate of colordevelopment is followed at 430 nm. Incubation temperature is 37° C.Activity is calculated from the slope of the rate curve as describedabove. A convenient range of enzyme concentrations for this method is0.001 to 0.02 units per cuvette.

One unit of cholesterol oxidase activity is that amount of enzymecatalyzing the production of 1 μ mole H₂ O₂ per minute at 37° C and pH7.0.

10. Determination of the Residual Cholesterol

Residual cholesterol is extracted from the fermentation broth and thecell suspension with a mixture of ethanol and n-heptane. Cholesterol inthe organic phase is silanized and measured by gas chromatography.

The following examples serve to illustrate particular embodiments of thepresent invention. Unless otherwise indicated, concentrations given inpercent are weight percent.

Nocardia cholesterolicum was grown in Fernbach flasks in the fivedifferent media described above under 2. Nutrient Media. Theextracellular as well as the intracellular, insoluble cholesteroloxidase produced in each medium was measured and the results arereported in Table 1.

The amount of intracellular, insoluble cholesterol oxidase was about thesame in the cells grown in the glycerol, glucose, and yeast extractmedia. The addition of inositol and yeast extract to the glycerol mediumresulted in the inhibition of the enzyme production. There was nointracellular, insoluble cholesterol oxidase activity in the cells grownin S-3 medium. Unexpectedly, however, there was a significant amount ofoxidase activity in the fermentation broth of this medium. In contrast,there was no extracellular activity in the broths obtained from themodified glycerol medium or from the yeast extract medium whereas thoseobtained from the glycerol medium or the glucose medium contained about20 - 30% of the total cholesterol oxidase activity.

Cholesterol oxidase produced by the cultures grown in the S-3 mediumreached a maximum value of 17.5 international units (U) per liter (FIG.1). This level is about 10 fold higher than that obtained with theglycerol medium. The amount of the extracellular enzyme decreased after48 hours. The cultures were efficient utilizers of cholesterol (morethan 90% of the cholesterol being used up in 3 days).

As the concentration of cholesterol in the medium diminished, the amountof the extracellular cholesterol oxidase decreased and the rate ofcholesterol utilization fell.

                  Table I                                                         ______________________________________                                        Effect of Medium on the Production                                            of Cholesterol Oxidase                                                        Cholesterol Oxidase U /Liter                                                           Intracellular                                                        Medium   insoluble     Extracellular                                                                             Total                                      ______________________________________                                        Glycerol 1.1           0.5         1.6                                        Modified                                                                      glycerol 0.4           0           0.4                                        Glucose  1.1           0.3         1.4                                        S-3      0             1.5         1.5                                        Yeast                                                                         extract  1.5           0           1.5                                        ______________________________________                                    

Description of Cholesterol Oxidase

To show that the extracellular cholesterol oxidase was functionally thesame as that extracted from the particulate fraction (i.e., theintracellular, insoluble fraction), its substrate specificity wasqualitatively determined. As shown in Table 2, the extracellular enzymedid not exhibit any activity with either β-sitosterol or ergosterol.However, it did not act upon cholestanol. There was no activity in theabsence of cholesterol or with boiled broth. In FIG. 2 it is seen thatthe extracellular cholesterol oxidase activity was dependent oncholesterol and deoxycholate (DOC) for its activity. All thesecharacteristics are identical to those of the enzyme obtained by thedetergent extraction of the particulate fraction (5).

These results indicate that medium was suitable for the production ofcholesterol oxidase since the yields were 10 fold higher and theextracellular enzyme appeared to be functionally similar to theintracellular insoluble cholesterol oxidase.

                  Table 2                                                         ______________________________________                                        Partial Characterization of the                                               Extracellular Cholesterol Oxidase                                             Enzyme                                                                        Source       Substrate      Activity*                                         ______________________________________                                        Unboiled broth                                                                             Cholesterol    +                                                 Boiled broth Cholesterol    -                                                 Unboiled broth                                                                             Nothing added  -                                                 Unboiled broth                                                                             β-Sitosterol                                                                            -                                                 Unboiled broth                                                                             Ergosterol     -                                                 Unboiled broth                                                                             Cholestanol    +                                                 ______________________________________                                         *+ = Presence of activity                                                     *- = Absence of activity                                                 

In the following examples unless otherwise noted the culture grown isNocardia cholesterolicum - rough stain and the medium used is the S-3medium described above.

EXAMPLE 2

Media similar to Nutrient Media 2. (d), above, but containing 0, 0.1,0.5, 5, and 10 g of S-3 per liter of medium were prepared, inoculatedand incubated in Fernbach flasks according to the general proceduregiven above. Each medium was assayed for cholesterol oxidase activityafter 24, 48, and 72 hours incubation time. The results are contained inTable 3.

These results indicate that the increase in the production ofcholesterol oxidase in S-3 medium is partly due to the richness of themedium and partly to the surfactant itself. For example, omission of S-3from the medium decreased the production of the enzyme by 50%. It isseen in Table 3 that S-3 is useful in the production of extracellularoxidase in the range from 2 to 10 g/1 of medium, preferably from 3 to 8g/1. S-3 is particularly desirable at a level of 4 to 6 g/1.

                  Table 3                                                         ______________________________________                                        Site and Amount of Cholesterol                                                Activity (U/l)*                                                               Conc.         Intra-    Intra-                                                of S-3                                                                              Time    cellular  cellular                                                                             Extra-                                         (g/l) (hrs)   Insoluble Soluble                                                                              cellular                                                                             Total                                   ______________________________________                                        0     24      3.8       --*    0      3.8                                           48      1.4       4.5    3.9    9.8                                           72      1.6       2.9    1.0    5.5                                     0.1   24      2.5       --*    0      2.5                                           48      --*       --*    4.1    4.1                                           72      2.1       2.2    1.3    5.6                                     0.5   24      2.6       --*    0      2.6                                           48      1.3       3.8    3.1    8.2                                           72      1.6       2.3    1.3    5.2                                     5.0   24      0.5       --*    0      0.5                                           48      1.3       3.1    17.2   21.6                                          72      1.4       1.8    14.0   17.2                                    10.0  24      0.2       --*    0      0.2                                           48      0         1.3    6.6    7.9                                           72      0         0.5    10.5   11.0                                    ______________________________________                                         *Not assayed.                                                            

EXAMPLE 3

Surfactants S-1, S-2, S-4, S-5, S-6, S-7 and S-8 were substituted forS-3 in the medium of Example 2 in a series of fermentations run in 250ml Erlenmeyer flasks using 25 ml of the medium of Example 2 per flask. Aflask containing S-3 as the surfactant was run concurrently as acontrol. The effects of three concentrations (0.5, 5.0 and 10.0 g/literof medium) of each of the surfactants on the level of extracellularcholesterol oxidase at 24, 48 and 72 hours were tested.

The optimum concentration of each surfactant and the correspondingfermentation time for maximum production of extracellular cholesteroloxidase are noted in Table 4. All of the surfactants tested increasedthe production of extracellular oxidase. In general, a concentration of5 g of detergent or less per liter was optimum for the production of theextracellular enzyme. With the exception of S-1, when the amount ofsurfactant was increased to 10 g per liter inhibition of enzymeproduction was observed.

                  Table 4                                                         ______________________________________                                                                 Extracellular                                                   Optimum Conc. Cholesterol                                                     of Surfactant Oxidase                                              Surfactant g/Liter       U./Liter                                             ______________________________________                                        S-1        10.0          4.4                                                  S-2        1.0           3.7                                                  S-4        5.0           9.8                                                  S-5        1.0           3.1                                                  S-6        5.0           4.6                                                  S-7        5.0           17.8                                                 S-8        1.0           4.5                                                  S-3        5.0           9.1                                                  No surfactant                                                                            --            1.6                                                  ______________________________________                                    

EXAMPLE 4 Effect of other Surfactants

Surfactants S-9, S-10, S-11, S-12, S-13 and S-14 - all of which containalkyl phenol groups were tested for their ability to improve the yieldof extracellular cholesterol oxidase. These compounds were tested inErlenmeyer flasks at three concentration levels (0.05, 0.5 and 5.0g/liter of medium) according to the procedure of Example 3. The optimumconcentration for each surfactant and the amount of extracellularcholesterol oxidase produced are shown in Table 5.

                  Table 5                                                         ______________________________________                                                                 Extracellular                                                   Optimum Conc. Cholesterol                                                     of Surfactant Oxidase                                              Surfactant g/Liter       U./Liter                                             ______________________________________                                        S-9        0.5           5.2                                                  S-10       0.5           3.9                                                  S-11       0.5           4.0                                                  S-12       0.5           6.8                                                  S-13       0.05          4.9                                                  S-14       0.05          2.4                                                  S-3        5.0           6.0                                                  No surfactant                                                                            --            3.0                                                  ______________________________________                                    

S-14 inhibited the production of the enzyme. The yields of cholesteroloxidase with S-10 and S-11 were slightly better than that observed inthe medium without any detergent. The production of the extracellularenzyme was significantly improved in the presence of S-9, S-12 and S-13.The levels of cholesterol oxidase obtained with these three surfactantswere comparable to those obtained with S-3. The concentrations of thesesurfactants, optimum for the production of cholesterol oxidase were 1 -2 orders of magnitude lower than the optimum concentration (5 g/liter)of S-3. These surfactants at concentrations above about 0.5 g per literinhibited the growth of Nocardia cholesterolicum.

EXAMPLE 5

A 150 liter fermenter containing 75 liters of sterilized modified S-3medium is inoculated with 6 liters of 19 hour old culture of Nocardiacholesterolicum. This inoculum is grown as described above. The mediumis agitated and aerated vigorously for 24 hours, preferably at astirring speed of 250 RPM and an air flow rate of 0.6 V.V.M. (volume ofair/volume of medium/ minute). The fermentation temperture is 30° C. Atthe end of 24 hours the cells are removed by centrifugation in arefrigerated continuous centrifuge. The centrifuged broth contains theextracellular cholesterol oxidase.

EXAMPLE 6 Effect of Culture

Various strains known to produce cholesterol oxidase were selected froman available culture collection. The strains were grown in S-3 mediumand the cholesterol oxidase produced at 24, 48 and 72 hours wasmeasured. Table 6 shows the maximum yield obtained.

                  Table 6                                                         ______________________________________                                        Production of Cholesterol Oxidase in S-3 Medium                                               Cholesterol Oxidase.sup.a                                                       Extra-    Intra-   U/liter                                  Strain            cellular  cellular Total                                    ______________________________________                                        Arthrobacter crystallopoites                                                                    0.9       --.sup.b --.sup.b                                 Arthrobacter strain NP                                                                          0.4       --.sup.b --.sup.b                                 Mycobacterium strain MA-7                                                                       0.6       0.0      0.6                                      Mycobacterium strain E-16                                                                       1.7       0.0      1.7                                      Mycobacterium rhodochrous                                                                       1.1       0.0      1.1                                      Corynebacterium species                                                                         4.0       0.0      4.0                                      Nocardia cholesterolicum-smooth                                                                 20.3      12.4     32.7                                     Nocardia cholesterolicum-rough                                                                  16.5      6.7      23.2                                     ______________________________________                                         .sup.a The figures represent the maximum production.                          .sup.b Not assayed.                                                      

EXAMPLE 7 Effect of Antifoam Polyglycol P-2000

Since the addition of a surfactant, such as for example S-3, is crucialto the production of extracellular cholesterol oxidase, it was necessaryto find means to control the excessive foaming owing to such addition.For this purpose, various concentrations of antifoam Polyglycol P-2000were studied. The results show that there is a slight increase in theamounts of the extracellular and total cholesterol oxidase with theincrease in the concentration of Polyglycol P-2000 up to 0.05 percent.In this concentration range (up to 0.05 percent), about 60 percent ofthe total enzyme was present extracellularly. Further increase in theconcentration of the antifoam caused inhibition of the enzyme synthesisand shifted the distribution of the enzyme in favor of the intracellularenzyme. Studies in a 150-liter fermenter showed that 0.03 percentPolyglycol P-2000 was adequate in controlling foaming owing to thepresence of surfactant S-3 in the medium.

EXAMPLE 8 Effect of Cholesterol

The effect of the amount of cholesterol present in the medium wasstudied by measuring the amount of enzyme produced with increasingconcentration of cholesterol. The results demonstrated a 13-foldincrease in the level of cholesterol oxidase produced as theconcentration of cholesterol was changed from 0 to 0.5%.

EXAMPLE 9 Effect of Inositol

The effect of inositol on the production of enzyme was examined, and itwas found that inositol inhibited the production of enzyme.

In view of the 3-fold increase in enzyme production realized withoutinositol, the experiments described in Examples 10-12 were performedutilizing a second modified S-3 nutrient medium as described below:

    ______________________________________                                        Modified S-3 Medium #2                                                        ______________________________________                                        Nutrient Broth   8.0 g                                                        S-3              5.0 g                                                        Yeast extract    1.0 g                                                        Cholesterol      1.0 g                                                        Distilled water  to 1 liger                                                   ______________________________________                                    

EXAMPLE 10 Testing of Other Steroids and Cholesterol Esters as Inducersof Cholesterol Oxidase

a. A number of steroids were tested for their ability to inducecholesterol oxidase. As shown in Table 7, the effectiveness of differentsteroids in inducing the enzyme varies markedly. Studies were made in250 ml Erlenmeyer flasks containing 25 ml of Modified S-3 Medium -2 asdescribed above. One gram of the steroid to be tested was added perliter instead of cholesterol. In this experiment, cholesterol induced anextracellular enzyme activity of 14.7 U per liter. Addition of groundmixed soy steroids to the medium resulted in the production of theenzyme, 65% of that induced by cholesterol. β-Sitosterol and5α-cholestan-3β-ol induced enzyme titres which were 65% and 59% of thoseinduced with cholesterol.

                  Table 7                                                         ______________________________________                                        Effectiveness of Other Steroids as                                            Inducers of Cholesterol Oxidase                                                             Extracellular                                                                 Activity Enzyme Activity                                                      (U/Liter)                                                                              % of Control.sup.b                                     ______________________________________                                        Cholesterol     14.7       100                                                β-Sitosterol                                                                             9.54       65                                                 Ground Mixed Soy Sterols                                                                      9.99       68                                                 5αCholestan-3β-ol                                                                  8.70       59                                                 Cholest-4-en-3-one                                                                            6.27       43                                                 Mixed Soy Steryl-2-                                                                           5.03       34                                                 Carbamato Glutaric Acid                                                       Potassium Salt                                                                7-Dehydrocholesterol                                                                          2.19       14                                                 ______________________________________                                         .sup.a Concentration of the sterols tested was 0.1%                           .sup.b The control medium contained cholesterol as the inducer.          

b. A total of 12 cholesterol esters were studied for their ability toinduce cholesterol oxidase. These were tested in the same manner as thesteroids. Cholesterol in the Modified, S-3 Medium #2 described above wassubstituted with the cholesterol ester to be investigated. Four of these12 esters of cholesterol studied, namely, linoleate, oleate, hexanoateand propionate, induced a level of cholesterol oxidase comparable tothat obtained with cholesterol in a similar medium (Table 8).Cholesteryl butyrate, cholesteryl decanoate and cholesteryl linolenatewere moderately successful in inducing the enzyme. The remaining estersinduced cholesterol oxidase to less than 50% of the control.Effectiveness of the esters with aromatic sidechains was even lower.

                  Table 8                                                         ______________________________________                                        Effectiveness of Cholesterol Esters as                                        Inducers of Cholesterol Oxidase                                                               Concentration                                                                             Enzyme Activity                                   Cholesterol Ester.sup.b                                                                       (m moles)   % of Control.sup.a                                ______________________________________                                        Cholesterol     2.6         100.0                                             Cholesteryl Propionate                                                                        2.3         79.7                                              Cholesteryl Butyrate                                                                          2.2         64.1                                              Cholesteryl Hexanoate                                                                         2.1         99.7                                              Cholesteryl Benzoate                                                                          2.0         45.0                                              Cholesteryl p-Nitrobenzoate                                                                   1.9         13.6                                              Cholesteryl Decanoate                                                                         1.9         63.0                                              Cholesteryl Laurate                                                                           1.8         54.2                                              Cholesteryl Myristate                                                                         1.7         49.9                                              Cholesteryl Palmitate                                                                         1.6         41.3                                              Cholesteryl Oleate                                                                            1.5         86.4                                              Cholesteryl Linoleate                                                                         1.5         112.0                                             Cholesteryl Linolenate                                                                        1.6         64.6                                              ______________________________________                                         .sup.a The control medium contained cholesterol as the inducer.               .sup.b The concentration of the esters was 0.1%                          

EXAMPLE 11 Effect of Yeast Extract

Omission of yeast extract from the Modified S-3 Medium -2 as describedabove reduced the levels of the extracellular enzyme by 50% (Table 9).The enzyme synthesis increased with increasing concentrations of yeastextract up to 0.15%. There was no further increase in the production ofthe enzyme with concentrations of yeast extract higher than 0.15%. Onthe contrary, there was repression of the enzyme in the medium with 1.0%yeast extract. The proportion of the enzyme released decreased from 81%to 26% when the concentration of yeast extract was varied from 0.0% to1.0%.

                  Table 9                                                         ______________________________________                                        Effect of Yeast Extract on the                                                Production of Cholesterol Oxidase                                             Concentration              Proportion                                         of Yeast     Extracellular of Enzyme                                          Extract      Enzyme %      Released                                           %            of Control.sup.a                                                                            %                                                  ______________________________________                                        0.0          50            81                                                 0.1          100           60                                                 0.15         16            41                                                 0.2          108           40                                                 0.5          113           40                                                 1.0          54            26                                                 ______________________________________                                         .sup.a The control medium contained 0.1% yeast extract.                  

EXAMPLE 12 Effect of Nutrient Broth Concentration

To determine an optimum concentration of Nutrient Broth the synthesis ofthe enzyme was carried out in Modified S-3 Medium -2 containing varyingconcentrations of this main nitrogen source. Table 10 illustrates therelative enzyme yield achieved with increasing concentration of NutrientBroth using 0.8% as a control.

                  Table 10                                                        ______________________________________                                        Effect of Nutrient Broth on the Production                                    of Cholesterol Oxidase                                                        Concentration of Relative Yield                                               Nutrient Broth   Extracellular                                                %                Cholesterol Oxidase                                          ______________________________________                                        0.0              18%                                                          0.2              50%                                                          0.4              80%                                                          0.8.sup.a        100%                                                         1.6              169%                                                         ______________________________________                                         .sup.a Control                                                           

EXAMPLE 13 Effect of Trace Salts

To determine the effect of trace salts the following salts were added toModified S-3 Medium -2:

    ______________________________________                                                         per liter                                                    ______________________________________                                        MgSO.sub.4. 7H.sub.2 O                                                                           125.0       mg                                             CaCl.sub.2 . 2H.sub.2 O                                                                          0.5         mg                                             FeSO.sub.4 . 7H.sub.2 O                                                                          40.0        mg                                             MnSO.sub.4 . H.sub.2 O                                                                           8.5         mg                                             ZnSO.sub.4 . 7H.sub.2 O                                                                          0.3         mg                                             NaCl               3.0         mg                                             ______________________________________                                    

A two-fold increase in the production of cholesterol oxidase was noted.

The results of the above-described examples indicated that the growthmedium for the production of the extracellular cholesterol oxidase fromNocardia cholesterolicum should contain as optimum concentrations, perliter: Nutrient Broth 8.0 g, Tween 40 5.0 g, yeast extract 1.5 g,cholesterol 5.0 g. However, scale-up studies in 150-liter fermenterindicated that cholesterol concentration should be about 1.0 g perliter. With this optimized medium up to 30 U of the extracellularcholesterol oxidase were produced.

While the invention has been described in detail with particularreference to preferred embodiments thereof, it will be understood thatvariations and modifications can be effected within the spirit and scopeof the invention.

We claim:
 1. In a method for the production of cholesterol oxidase bygrowing a cholesterol oxidase producing micro-organism in a mediumcomprising a cholesterol oxidase inducer, the improvement wherein saidcholesterol oxidase inducer is β-sitosterol.
 2. In a method for theproduction of cholesterol oxidase by growing a cholesterol oxidaseproducing micro-organism in a medium comprising a cholesterol oxidaseinducer, the improvement wherein said cholesterol oxidase inducer is aground mixed soy sterol.